Detection of copy number variations in pediatric ETV6/RUNX1-positive acute lymphoblastic leukemia with multiplex ligation-dependent probe amplification / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the application of multiplex ligation-dependent probe amplification (MLPA) in the detection of copy number variations (CNVs) in pediatric ETV6/RUNX1-positive acute lymphoblastic leukemia (ALL), to compare this method with conventional karyotype analysis and fluorescence in situ hybridization (FISH), and to evaluate the value of MLPA.</p><p><b>METHODS</b>The clinical data of 95 children with ETV6/RUNX1-positive ALL who were treated from January 2006 to November 2012 were analyzed retrospectively, including clinical features, results of karyotype analysis, and results of FISH. CNVs were detected with MLPA.</p><p><b>RESULTS</b>CNVs were detected in 73 (77%), and the median number of CNVs was 1 (range 0-6). The CNVs of EBF1, CDKN2A/2B, PAX5, ETV6, RB1, and BTG1 were detected in more than 10% of all the patients. The changes in the chromosome segments carrying the genes with CNVs detected by MLPA were not detected by conventional karyotype analysis. The coincidence rate between the CNVs in ETV6 gene detected by FISH and those detected by MLPA was 66%.</p><p><b>CONCLUSIONS</b>MLPA is an efficient and convenient method to detect CNVs in children with ETV6/RUNX1-positive ALL.</p>

Significance of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia without reproducible chromosomal abnormalities / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To identify the incidence of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia (B-ALL) without reproducible chromosomal abnormalities and to investigate the association between PAX5 abnormalities and prognosis of ALL.</p><p><b>METHODS</b>Multiplex ligation-dependent probe amplification was used to determine the copy numbers of PAX5 gene in children newly diagnosed with B-ALL without reproducible chromosomal abnormalities between April 2008 and April 2013 and controls (children with non-hematologic diseases or tumors). The patients were classifiied into deletion group and non-deletion group based on the presence of PAX5 deletion.</p><p><b>RESULTS</b>Eighteen (21%) out of 86 children with B-ALL had PAX5 deletion. The deletion group had a significantly higher total white blood cell count at diagnosis than the non-deletion group (P=0.001). The Kaplan-Meier analysis demonstrated that the deletion group had a significantly lower disease-free survival (DFS) rate than the non-deletion group (0.69±0.12 vs 0.90±0.04; P=0.017), but there was no significant difference in the overall survival rate between the two groups (P=0.128). The Cox analysis showed that PAX5 deletion was a risk factor for DFS (P=0.03).</p><p><b>CONCLUSIONS</b>PAX5 deletion is an independent risk factor for DFS in B-ALL children without reproducible chromosomal abnormalities.</p>

Association between clinical outcome and gene mutation in children with Fanconi anemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the association between clinical outcome and gene mutations in children with Fanconi anemia (FA).</p><p><b>METHODS</b>A retrospective analysis was performed for the clinical data of six children with the same severity of FA and receiving the same treatment. At first, single cell gel electrophoresis and chromosome breakage induced by mitomycin C were performed for diagnosis. Then the gene detection kit for congenital bone marrow failure diseases or complementation test was used for genotyping of FA. Finally the association between the clinical outcome at 3, 6, 9, or 12 months after treatment and gene mutation was analyzed.</p><p><b>RESULTS</b>Of all the six FA children, five had FANCA type disease, and one had FANCM type disease; four children carried two or more FA gene mutations. Among the children with the same severity of FA, those with more FA mutations had a younger age of onset and poorer response to medication, and tended to progress to a severe type.</p><p><b>CONCLUSIONS</b>Children carrying more than two FA mutations have a poor clinical outcome, and hematopoietic stem cell transplantation should be performed as soon as possible.</p>

Methylation of the genes in the 9P21 region in children with acute myeloid leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the methylation rate of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase inhibitor 2B (CDKN2B) in the 9P21 region in children with acute myeloid leukemia (AML) and the association of gene methylation with clinical features and outcomes.</p><p><b>METHODS</b>The clinical data of 58 children who were newly diagnosed with AML between January 2010 and December 2012 were retrospectively analyzed. Thirty-eight healthy children were recruited as the control group. Genomic DNA was extracted from bone marrow or peripheral blood of the 58 patients and 38 healthy children. The methylation status of CDKN2A and CDKN2B was analyzed by methylation-specific multiplex ligation-dependent probe amplification.</p><p><b>RESULTS</b>Gene methylation was not found in healthy children. Methylation probes of 44 patients were detected in 58 patients. The methylation of CDKN2A was detected with 136 bp and 237 bp methylation probes. The methylation of CDKN2B was detected with 130 bp, 210 bp, 220 bp, and 417 bp methylation probes. The methylation rate of CDKN2A was 5%, while the methylation rate of CDKN2B was 76%. The methylation detected by some probes was associated with sex, hemoglobin, and platelet count at the first visit.</p><p><b>CONCLUSIONS</b>The methylation of CDKN2B is a common event in children with AML, while the methylation of CDKN2A is relatively rare.</p>

Significance of IKZF1 gene copy number abnormalities in BCR/ABL-negative B-lineage acute lymphoblastic leukemia in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To identify IKZF1 gene copy number abnormalities in BCR/ABL-negative B-lineage acute lymphoblastic leukemia (B-ALL) in children, and to investigate the association between such abnormalities and prognosis.</p><p><b>METHODS</b>Multiplex ligation-dependent probe amplification (MLPA) was applied to detect IKZF1 gene copy number abnormalities in 180 children diagnosed with BCR/ABL-negative B-ALL. These children were classified into IKZF1 deletion group and IKZF1 normal group according to the presence or absence of IKZF1 gene deletion. The association between IKZF1 copy number abnormalities and prognosis of children with BCR/ABL-negative B-ALL was analyzed retrospectively.</p><p><b>RESULTS</b>Among 180 children, 27 (15.0%) had IKZF1 deletion; among the 27 children, 4 had complete deletions of 8 exons of IKZF1 gene, 17 had deletion of exon 1, 3 had deletions of exons 4-7, and 3 children had deletions of exons 2-7. Compared with those in the IKZF1 normal group, children in the IKZF1 deletion group had higher white blood cell (WBC) count and percentage of individuals with high risk of minimal residual disease at the first visit. IKZF1 deletions often occurred in BCR/ABL-negative children with no special fusion gene abnormalities. They were frequently accompanied by abnormalities in chromosomes 11, 8, 5, 7, and 21. The analysis with Kaplan-Meier method showed that disease-free survival (DFS) in the IKZF1 deletion group was significantly lower than that in the IKZF1 normal group (0.740 ± 0.096 vs 0.905 ± 0.034; P=0.002). Cox analysis showed that after exclusion of sex, age, initial WBC count, cerebrospinal fluid state at the first visit, prednisone response, and chromosome karyotype, IKZF1 deletion still affected the children's DFS (P<0.05).</p><p><b>CONCLUSIONS</b>Some children with BCR/ABL-negative B-ALL have IKZF1 deletion, and IKZF1 deletion is an independent risk factor for DFS in children with BCR/ABL-negative B-ALL.</p>

Efficacy and safety of imatinib for the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia in children / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the efficacy and safety of Chinese Childhood Leukemia Group ALL 2008 (CCLG-ALL2008) protocol combined with tyrosine kinase inhibitor (TKI, imatinib) for the treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) in children.</p><p><b>METHODS</b>The clinical data of 53 patients aged less than 15 years when first diagnosed with Ph+ ALL between October 2008 and December 2013 were retrospectively analyzed. The patients were assigned to two groups: HR (n=26) and HR+TKI (n=27). The HR group was treated with CCLG-ALL2008 protocol (for high-risk patients). The HR+TKI group was treated with imatinib in combination with CCLG-ALL2008 protocol (for high-risk patients).</p><p><b>RESULTS</b>The complete remission rate and chemotherapy induction-related mortality rate in the TKI+HR and HR groups were 100% vs 75% and 0 vs 15%, respectively. The 3-year event-free survival (EFS) rate in the HR group was (6±5)%; the 5-year EFS rate of the TKI+HR group was (52±11)%. Compared with the HR group, the TKI+HR group had no increase in the toxic responses to chemotherapy and had a decrease in the infection rate during the induction period.</p><p><b>CONCLUSIONS</b>Application of imatinib significantly improves the clinical efficacy in children with Ph+ ALL and has good safety.</p>

Genotype analysis and telomere length measure in patients with dyskeratosis congenita / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To analysze genotype and measure telomere length in two Chinese patients with dyskeratosis congenita(DC).</p><p><b>METHODS</b>The peripleral blood DNA was extracted in two patients characterized by mucocutaneous abnormalities (abnormal nails, lacy reticulated skin pigmentation, and oral leukoplakia), bone marrow failure, DC genes were amplified by polymerase chain reaction (PCR), including DKC1, TERT, TERC, TINF2, NOP10, NHP2, then DNA sequencing was performed for abnormal exons. Lymphocyte telomere length was measured by flow cytometry-fluorescence in situ hybridization(Flow-FISH).</p><p><b>RESULTS</b>Abnormal peaks were found in exon 6 of TINF2 gene of the two patients and a 811C→T transition in TINF2 gene in one patient. DNA sequencing showed a 848C→A transition in TINF2 gene in another patient. Relative telomere length was remarkable less than that of normal children with same age.</p><p><b>CONCLUSIONS</b>Physician should think about DC if the young patients with mucocutaneous abnormalities and marrow failure. Early detection of related genes and measurernant of tolomere length may contribute to avoid misdiagnosis. TINF2 c.811C→T (Q271X) and TINF2 c.848C→A (P283H) exist in the two patients, it is reported in China for the first time.</p>

Copy number variations in pediatric acute myeloid leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To evaluate the copy number variations (CNV) of gene in pediatric acute myeloid leukemia (AML) and its correlation with clinical features and prognosis.</p><p><b>METHODS</b>The clinical data of 130 children aged <14 years with newly diagnosed AML from May 2006 to March 2013 were analyzed restrospectively. The CNV were analyzed by multiplex ligation-dependent probe amplification (MLPA). Thirty-eight normal children were selected in control group. All the data were statistically analyzed using SPSS16.0 software.</p><p><b>RESULTS</b>gene CNV of 2p24.3(MYCN), 10q23(PTEN) and 13q14(RB1, MIR15A, DLEU) were detected in more than 10% of the patients. CNV were detected in 49 cases(37.7%). The median loss and gain CNV frequencies per sample were 4. The CNV of TP53 correlated significantly with relapse. The loss ond gain CNV have no influence to EFS, DSF and OS. CNV were detected in the twelve percent of patients, but they were not detected with routine karyotype method.</p><p><b>CONCLUSION</b>The MLPA technique combined with karyotyping makes a substantial increase in the diagnostic rate. Patients with TP53 alterations have significantly higher relapse rate.</p>

Long-term efficacy of CAMSBDH-ALL chemotherapy protocol for the treatment of childhood acute lymphoblastic leukemia / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the long-term efficacy of CAMSBDH-ALL chemotherapy protocol for the treatment of childhood acute lymphoblastic leukemia (ALL).</p><p><b>METHODS</b>Three hundred and eighteen children who were newly diagnosed with ALL between January 1999 and December 2007 were enrolled in this study. Among the 318 children, 83 children who hospitalized before December 2002 were treated with CAMSBDH-ALL99 protocol, including 48 patients of standard risk and 35 patients of high risk. The patients (n=235; 131 in standard risk and 104 in high risk) who hospitalized after December 2002 were treated with CAMSBDH-ALL03 protocol. Patients in the CAMSBDH-ALL99 protocol group were treated with conventional chemotherapy. CAMSBDH-ALL03 protocol was modified based on the CAMSBDH-ALL99 protocol.</p><p><b>RESULTS</b>The long-term overall survival (OS) and event-free-survival (EFS) in the CAMSBDH-ALL03 group was significantly higher than in the CAMSBDH-ALL99 (P<0.01). The long-term OS and EFS of standard risk and high risk patients in the CAMSBDH-ALL03 protocol group were significantly higher than in the CAMSBDH-ALL99 protocol group (P<0.01). The CAMSBDH-ALL03 protocol group showed a significantly lower recurrence rate (28.9%) than in the CAMSBDH-ALL99 protocol group (50.6%) (P<0.05). The mortality rate in the CAMSBDH-ALL03 protocol group was 28.5% vs 56.6% in the CAMSBDH-ALL99 protocol group (P<0.05).</p><p><b>CONCLUSIONS</b>The therapeutic effect of the CAMSBDH-ALL03 protocol is supior to the CAMSBDH-ALL99 protocol group for childhood ALL, with a higher long-term survival rate.</p>

Correlation of single-cell gel electrophoresis and mitomycin C-induced chromosomal breakage for chromosomal instabiligy in children with Fanconi anemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Fanconi anemia (FA) is characterized by bone marrow failure, congenital abnormalities and predisposition to neoplasia. Hypersensitivity of FA cells to the clastogenic effect of mitomycin C (MMC) provides a unique marker for the diagnosis before the beginning of hematological manifestations. The aim of this study was to evaluate the relationship between Single-Cell Gel Electrophoresis (SCGE) and mitomycin C-induced chromosomal breakage in children with FA.</p><p><b>METHOD</b>Between January 2007 and June 2011, 248 children (< 15 years) with hypocytosis were included. Chromosomal breakage was induced by MMC 0 ng/ml, 40 ng/ml, and 80 ng/ml. SCGE was performed at the same time. We analyzed the results of the two methods and compared with each other. The receiver operating characteristic (ROC) curve was used to evaluate the parameters in SCGE.</p><p><b>RESULT</b>Seventeen patients were diagnosed as FA and 231 as non-FA. Chromosomal breakage was found to be significantly higher in FA patients [(32.2 ± 4.8)%] than non-FA [(19.9 ± 3.0)%] and controls[(21.6 ± 4.8)%] when induced by MMC 80 ng/ml. The parameters of SCGE were significantly different between FA patients and non-FA or controls. All the parameters were rectilinearly correlated with MMC (P = 0.000). The most closely correlated parameter was the rate of comet cell (r = 0.848, P = 0.000). The results of ROC curves suggested the comet cell rate (0.999) was more important.</p><p><b>CONCLUSION</b>SCGE might be used to discriminate between FA and non-FA individuals. The relationship between SCGE and MMC-induced chromosomal breakage was significant. The rate of comet cell was the important parameter.</p>

FLT3 mutations in children with acute myeloid leukemia: a single center study / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the clinical significance of FMS-like tyrosine kinase 3 (FLT3) mutations including internal tandem duplication (ITD) mutation and point mutation of tyrosine kinase domain (TKD) in children with acute myeloid leukemia (AML).</p><p><b>METHODS</b>Bone marrow samples from 116 children with newly-diagnosed AML were obtained. Gene mutations of FLT3/ITD and FLT3/TKD were detected by RT-PCR. The relationship of FLT3 gene mutations with the clinical characteristics and the therapeutic efficacy was observed.</p><p><b>RESULTS</b>FLT3/ITD and FLT3/TKD mutations were detected in 9 cases (7.8%) and 13 cases (11.2%) respectively out of the 116 children. FLT3/ITD mutations were observed in 3 cases of AML-M3 (3/9; 33.3%) and in 3 cases of AML-M5 (3/9; 33.3%). FLT3/TKD mutations were the most common in AML-M3 patients (10/13; 76.9%). The patients with FLT3/ITD mutations had a significantly higher peripheral WBC count and marrow blast percentage compared with the patients without FLT3/ITD mutations at diagnosis (P<0.01). The 3-year overall survival rate in patients with FLT3/ITD mutations was significantly lower than that in patients without FLT3/ITD mutations (38.9% vs 64.3%; P<0.05).</p><p><b>CONCLUSIONS</b>FLT3/TKD mutations are common in children with AML-M3. The AML children with FLT3/ITD mutations present a high peripheral WBC count and a high marrow blast percentage at diagnosis and have an unfavorable outcome.</p>